Sphingolipids are emerging as key regulators of stress responses in mammalian and yeast cells. In yeast, heat stress causes an acute upregulation of sphingolipid synthesis. In mutant yeast strains where this upregulation does not occur, the heat stress response is impaired, thus, we hypothesize that sphingolipids mediate some components of the total heat stress response. The major outcome of the heat stress response is adaptation to heat, which is achieved through dramatic changes in gene regulation. Indeed, greater than 30% of the yeast genome is regulated in response to heat. Preliminary studies using microarray hybridization indicate that when heat-induced sphingolipid generation is blocked greater than 10% of the expected changes in gene regulation do not occur. The proposed research will further identify sphingolipid-dependent gene regulatory events, address mechanisms of sphingolipid-induced 'transcriptional regulation and also attempt to identify specific sphingolipids responsible for changes in gene regulation. In order to accomplish this, we propose to use a combined microarray and molecular genetic approach to determine novel sphingolipid dependent transcriptional events and signaling targets.